The present invention relates to the field of diagnosis. In particular, it relates to a method for producing polybiotinylated dendrimer compounds useful for signal amplification, in particular during the detection of an analyte in a diagnostic test.
Probes which allow the detection of analytes such as proteins and nucleic acids are widely used and are tools of choice for in vitro and in vivo diagnosis. However, it may be difficult to detect these analytes because they are not always present in sufficiently large quantities. A detection amplification system is therefore necessary.
Several techniques have been developed for detecting analytes in insufficient quantities. These techniques involve either increasing the quantity of analyte, for example with PCR or NASBA techniques, or amplifying the detection, for example by using structures having multiple markers.
Thus, patent application EP0774119A describes conjugates having multiple markers comprising a polymer support having a maximum of 100 monomer units, which contains 1 to 10 molecules of haptene and 1 to 10 marker groups, and also the preparation of these conjugates by solid-phase synthesis (a) by introducing, at predetermined positions of the support, monomer derivatives which are coupled covalently to haptene molecules and/or marker groups and/or (b) by coupling haptene molecules and/or marker groups to reactive groups of the support, after the synthesis.
Dendrimers are clearly defined macromolecules which have multiple branches and which terminate in multiple peripheral functional groups. One of the advantages of dendrimers, due to the fact that they have multiple functional groups, is that they can be used as stable coupling agents, as described in patent application WO03/72546 filed by the Applicant. Another advantage also resides in the fact that they are structures with multiple markers because their peripheral functional groups can be used to bind multiple markers such as biotin, fluorophores or a combination thereof. Thus, patent application WO01/02861 describes dendrimers bound on the one hand to 1 to 1200 probes and on the other hand to 1 to 1200 markers. These dendrimers are used for signal amplification.
Other polybiotinylated dendrimers have also been described in patent application WO00/72802.
However, conventional methods for preparing dendrimers have the drawbacks that the dendrimers are synthesised in liquid phase, which is more complex and more lengthy than in solid phase. Furthermore, some methods produce non-neutral and non-polarised dendrimers.